Difference between revisions of "Team:Aix-Marseille/Protocols"
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{{Aix-Marseille|title=Protocols}} | {{Aix-Marseille|title=Protocols}} | ||
| − | === | + | ===DH5α competent cells preparation=== |
#The day before the preparation, make overnight starters with 100 µL of bacterial cells diluted in 5 mL of LB in a 50 mL Erlenmeyer.<br> | #The day before the preparation, make overnight starters with 100 µL of bacterial cells diluted in 5 mL of LB in a 50 mL Erlenmeyer.<br> | ||
#Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and 50 mL Falcon tubes, and p1000 tips at -80°C for future use.<br> | #Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and 50 mL Falcon tubes, and p1000 tips at -80°C for future use.<br> | ||
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|- | |- | ||
| KCl (1M) | | KCl (1M) | ||
| − | | 400 | + | | 400 µL |
|- | |- | ||
| MOPS (1M) | | MOPS (1M) | ||
| − | | 200 | + | | 200 µL |
|- | |- | ||
| CaCl2 (0.1M) | | CaCl2 (0.1M) | ||
| Line 52: | Line 52: | ||
|- | |- | ||
| Glycerol (80%) | | Glycerol (80%) | ||
| − | | 750 | + | | 750 µL |
|- | |- | ||
| H2O | | H2O | ||
| − | | 250 | + | | 250 µL |
|} | |} | ||
Revision as of 19:53, 13 August 2018
Protocols
DH5α competent cells preparation
- The day before the preparation, make overnight starters with 100 µL of bacterial cells diluted in 5 mL of LB in a 50 mL Erlenmeyer.
- Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and 50 mL Falcon tubes, and p1000 tips at -80°C for future use.
- The next day, recover your starters and seed 1/60 bacterial cells in 200 mL of LB, then incubate at 37°C and 180 rpm.
- Keep measuring the OD600. When it's between 0,4 and 0,6, recover your cells an pool them in cold 50 mL Falcon tubes.
- Centrifuge tubes at 4000 rpm and 4°C for 10 minutes.
- Discard supernatant and resuspend each Falcon tube in 20 mL of buffer 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.
- Discard supernatant and resuspend each Falcon tube in 8 mL of buffer 2.
- Take 100 µL aliquots in Eppendorf tubes and stock them at -80°C.
| Product | For 40 mL |
|---|---|
| KAc (1M) | 1.2 mL |
| MnCl2 (0.5M) | 4 mL |
| KCl (1M) | 4 mL |
| CaCl2 (0.1M) | 4 mL |
| Glycerol (80%) | 7.5 mL |
| H2O | 19.3 mL |
| Product | For 8 mL |
|---|---|
| KCl (1M) | 400 µL |
| MOPS (1M) | 200 µL |
| CaCl2 (0.1M) | 3 mL |
| Glycerol (80%) | 750 µL |
| H2O | 250 µL |